Alternaria alternata is the main pathogenic fungus of postharvest black spots in fruits and vegetables. This study aimed to explore the antifungal activity of methionine on
A. alternata in vitro and to reveal related antifungal mechanisms through a metabolomics analysis. The results showed that the inhibitory effects of L-methionine (Met) treatment on mycelium growth, spore germination, and the germ tube elongation of
A. alternata were enhanced with an increase in the Met concentration, but the inhibitory effects decreased when the Met concentration was higher than 50 mmolL
−1. The results of propidium iodide staining and scanning electron microscopy showed that the Met treatment damaged the plasma membrane integrity of the
A. alternata spores and caused an irreversible deformation of mycelium. In addition, after the Met treatment, the leakage of electrolytes, nucleic acid, and proteins in the
A. alternata cells was significantly higher than that in the control group, indicating that the Met treatment increased the permeability of the cell membranes. Eighty-one different metabolites, divided into seven categories, were identified through the metabolomics analysis, including forty-three downregulated metabolites and thirty-eight upregulated metabolites. Among them, these differential metabolites were mainly involved in amino acid synthesis and metabolism, the pentose phosphate pathway, and the TCA cycle. Therefore, the antifungal effect of the Met treatment on
A. alternata was mainly to damage the integrity of the cell membranes, make nucleic acid and protein contents leak, and affect the TCA cycle, carbohydrate metabolism, amino acid synthesis metabolism, and the metabolic pathways associated with cell membrane biosynthesis. Thus, the growth and development of
A. alternata were inhibited. The research enriched the investigation of the effect of the antifungal mechanism of Met treatment on
A. alternata and provided a theoretical basis for the application of Met to prevent and treat postharvest black spots in fruits and vegetables.
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